The present invention relates to a method for performing a qualitative or quantitative assay which may be used for detecting quantifying or monitoring oxidative damage especially in relation to an inflammatory condition. Detection of nitrated specific proteins or fragments thereof versus equivalent non-nitrated proteins or fragments thereof, may serve as an index for oxidative damage in for example inflammatory bowel disease, systemic lupus erythematosus, arthritis, cancer, Parkinson's or Alzheimer's disease.
Throughout a lifetime organisms are challenged with numerous events and conditions that generate reactive oxygen species (ROS). An increase in an organism's rate of ROS production or a decrease in their rate of scavenging will increase the oxidative modification of cellular molecules, including DNA and proteins. Oxidation may have deleterious effects on protein function and stability. Many enzymes have been shown to lose their biological activity as a consequence of oxidation. Other effects of oxidation are lowered temperature-stability and changes in protein susceptibility towards proteolysis; the latter might lead to an accumulation of oxidized proteins unable to undergo degradation. Protein oxidation may be implicated in the pathogenesis of several diseases such as neurodegenerative diseases, cancers, arteriosclerosis, cataractogenisis, dysplasia, dystrophia and inflammatory diseases as well as in normal ageing.
Some of the common ROS generating processes and systems known to modify proteins are irradiation, inflammation, metal catalysed reactions such as Fe(II) or Cu(I) reduction and various other oxidizing compounds or free radicals, including nitric oxide (NO), peroxy-itrite, H2O2 or hydroxyl, hydroperoxyl superoxide and lipid peroxyl radicals. A number of ROS are formed by specific enzyme systems such as nitric oxide synthetase (NOS), cyclo-oxygenase and mono-amine oxidase B, whereof some are induced under inflammatory conditions.
Under normal conditions the oxidative potential in the micro-environment of an organism is under tight control by a number of balancing systems including antioxidants, free radical scavengers, reductases, peroxidases, catalase, glutathione-S-transferase, super-oxide dismutase and various metal-binding proteins. These systems can be viewed as protection mechanisms, more than repair systems. Actual repair mechanisms specific for oxidative damaged proteins are rare, whereas the oxidative damage to nucleic acids is subject to highly efficient repair systems (Stadtman and Levine 2000).
Nitric oxide (NO) is produced in many tissues and regulates diverse functions, such as smooth muscle relaxation, non-specific defense against microorganisms, neurotransmitter and a possible modulator of the cartilage matrix. Nitric oxide synthetase (NOS) is responsible for the production of NO. There are two classes of NOS, a constitutive (cNOS) and an inducible (iNOS) form. iNOS activity appears in response to various cytokines, and produces a much larger amount of NO than cNOS. iNOS activity is thought to account for the proinflammatory effects of NO, as seen in conditions such as inflammatory bowel diseases, spontaneous gut inflammation, cardiovascular inflammation and arthritic diseases (osteoarthritis (OA) or rheumatoid arthritis (RA)).
The large cytotoxicity of NO is partly due to its ability to react with superoxide anion (O2−) to generate peroxynitrite anion (ONOO−) and its conjugate acid, peroxynitrous acid (ONOOH). At neutral pH ONOO− is partly protonated, generating ONOOH, which rapidly decomposes to nitrate. These strong oxidants might seriously compromise cellular regulation, as it is capable of nitrating aromatic compounds like free phenylalanine, tyrosine and tryptophan as well as peptide chains containing these amino acids. This result in nitrophenylalanine, nitro-tryptophan and nitrotyrosine, the later can also be generated through the combined hydroxylation and nitration of a phenylalanine residue (Lin et al 2000). The nitration is irreversible and inhibits the phosphorylation of tyrosine and tryptophan residues, thus interfering with signal transduction pathways.
In OA and RA, NO is produced in large amounts by chondrocytes, macrophages and inflamed synovium. A high level of nitrite/nitrate has been found in the synovial fluid, serum and urine of patients with OA and RA (Lotz 1999). However, elevated NO levels cannot be considered a specific marker for any given disease or condition, as several different processes and tissues can give rise to systemic elevated NO levels.
The major clinical manifestation of RA as well as OA is an abnormal and degraded cartilage. However, until now it has been difficult to directly assess the ongoing cartilage destruction in arthritis patients, because specific markers for this process have not been available in the clinical practice. At clinical diagnosis of OA and RA, damage to cartilage in joints is recorded by X-ray, which reveals a loss of joint space as cartilage is destroyed and lost. Furthermore the patients are scored according to the pain and mobility problems caused by the joint destruction, but even though a number of standardised rating systems have been introduced, it is difficult to quantify these parameters. Other markers used for assessment of RA patients, such as C-reactive protein and Rheumatoid factors are associated with the inflammatory process involved in the disease, but are probably not directly related to the level of cartilage destruction and they are not specific for RA.
Detection of metabolites, such as cartilage oligomeric matrix protein (COMP), hyaluronates, aggrecan and collagen type II or III fragments arising from destruction of joints affected by inflammatory disease have been reported (Moller 1998, Wollheim 1996, U.S. Pat. No. 5,919,634, U.S. Pat. No. 6,132,976 and PCT application WO 01/38872). The clinical usefulness of these markers, however, remains to be proven.
The detection of NO2-modified amino acids is known from the PCT patent applications WO 96/04311 and WO 98/29452. These patent applications disclose the sequence independent detection of a nitrotyrosine or a nitro-tryptophan residue in a protein or in its free form using an antibody, which specifically recognizes the nitro-group. Such an antibody might be used to assess a pathological condition relating to an abnormal level of nitrotyrosine. However the antibody will not be able to assess the problem in relation to a specific tissue or protein as it recognizes nitrotyrosine independent of the surrounding amino acid sequence.
The present invention relates to methods for quantifying NO2-modified amino acids within the specific context of a protein or fragments thereof. Determination of such nitrated specific proteins or fragments thereof enables an assessment of oxidative damage and metabolic state of the given protein. This provides diagnostic assays, which associate metabolic changes and oxidative damage in specific diseases.
According to the present invention there is provided a method for performing a qualitative or quantitative assay for protein oxidation, comprising detecting in a sample an amino acid sequence which is characteristic of a specific protein and which contains one or more aromatic amino acid residues in nitrated form. This includes a method for detecting oxidative damage in a mammal. The method may comprise monitoring oxidative damage by detecting one or more nitrated aromatic residues in combination with an amino acid sequence specific to a protein or peptide, preferably specific to a certain tissue, in a biological sample. Preferred aromatic amino acid residues are nitrotyrosine and/or nitrotryptophan.
The method of the present invention can be applied for monitoring a pathological process involving an oxidative damage correlating with non-inflammatory diseases like arteriosclerosis, cancer, Alzheimer's disease, Parkinson's disease or inflammatory diseases like asthma, cardiovascular inflammation, diabetes, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, arthritis.
The method of the present invention will enable the monitoring of a catabolic process of a joint tissue by measuring a protein or peptide derived from the extra cellular matrix of cartilage, joint synovium or subchondral bone. Such a protein might be collagen types I, II, III, VI, IX or XI, aggrecan, biglycan, chondromodulin, cartilage link protein, cartilage oligomeric matrix protein, cartilage intermediate layer protein or a fragment thereof, wherein a nitrotyrosine and/or nitrotryptophan residue is located.
The detection performed in the method of the present invention may be carried out using an antibody, which specifically binds a nitrated epitope comprising at least one nitrated aromatic amino acid residue in conjunction with a specific amino acid sequence.
The detection performed in the method of the present invention can also be carried out utilizing two antibodies, one which is specific for the nitrated aromatic amino acid residue in a context independent manner and one which recognizes the specific amino acid sequence.
The detection methods of the present invention can be performed in a way that generate an index of oxidative damage/inflammation, by using a second antibody, which specifically binds a non-nitrated amino acid sequence equivalent to the nitrated sequence bound by the antibody which recognizes the nitrated epitope described above, thereby generating a ratio between a specific nitrated and non-nitrated protein or peptide.
Alternatively, the relative amounts of nitrated and non-nitrated protein or peptide may be differently expressed, for instance as a difference or as the ratio of the nitrated content to the total of nitrated and non-nitrated content.